DNA detection method

ABSTRACT

Picogram amounts of DNA can be detected in a sample by the use of high-affinity, single-stranded DNA binding proteins. The assay is applicable not only to pure DNA samples but also to samples containing significant amounts of protein.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional of application Ser. No. 285,895, filed Dec. 15,1988, which is a continuation-in-part of U.S. patent application Ser.No. 093,361, filed Sept. 4, 1987.

This Application is a continuation in part of U.S. application Ser. No.093,361 filed Sept. 4, 1987, which disclosure is here by incorparated byreference.

INTRODUCTION

1. Technical Field

This invention relates to methods for detecting the presence of DNA. Themethod employs high affinity binding proteins for single stranded DNA.

2. Background

The amount of DNA in a sample has traditionally been measured either byspectrophotometric means or fluorometrically with the use of ethidiumbromide. If amounts of contaminants such as other nucleic acids,protein, phenol, or agarose) the spectrophotometric measurement of theamount of ultraviolet (UV) irradiation absorbed is simple and accurate.However, if there is contamination with protein or compounds whichabsorb strongly in the UV, such as phenol, accurate quantitation of theamount of DNA will not be possible. Furthermore, this technique is onlysuitable for samples containing DNA in the μg/ml range.

If the amount of DNA in the sample is small, or if the sample containssignificant quantites of impurities, the amount of DNA may be estimatedfrom the intensity of UV-induced fluorescence emitted by ethidiumbromide intercalated into the DNA. The amount of fluorescence isproportional to the total amount of DNA. The quantity of DNA in thesample therefore can be estimated by comparing the fluorescent yield ofthe sample with that of a series of standards. As little as 1 to 5 μg/mlof DNA can be detected by this method. With the use of amini-fluorometer (such as that manufactured by Hoefer ScientificInstruments, San Francisco, Calif.) and the fluorochrome Hoechst 33258,the sensitivity may be increased to 10 ng/ml.

With the advent of recombinant DNA technology, it has become imperativeto be able to identify significantly lower concentrations of DNA in asample, for example, any contaminating DNA which may be present in arecombinant product. The contaminating DNA may be non-specific and ofunknown sequence. Therefore, enzyme amplification of sample DNA (usingfor example the DNA polymerase chain reaction method) is difficult forlack of universal primers for DNA synthesis. There is, therefore,substantial interest in being able to detect rapidly and accurately thepresence of extremely small amounts of DNA.

RELEVANT LITERATURE

Krauss et al., Biochemistry (1981) 22:5346-5352 disclose the binding ofsingle-stranded binding proteins from E. coli to oligonucleotides.Vogelstein and Gillespie, Proc. Natl. Acad. Sci. USA (1979) 76:615-619disclose the binding of DNA to glass. Kung et al. disclose thepurification of topoisomerase I from Micrococcus luteus by high saltelution from a DNA-sepharose column; J. Biol. Chem. (1977)252:5398-5402. The following are review articles pertaining to DNAbinding proteins. Gellert, The Enzymes, Vol. XIV (1981) 345-366; Wang,The Enzymes, Vol. XIV (1981) 332-343; Chase, Ann. Rev. Biochem. (1986)55:103-136; Kowalczykowski et al., The Enzymes, Vol. XIV (1981) 75-444.

SUMMARY OF THE INVENTION

A novel method is provided for detecting the presence of DNA in asample, which employs a DNA-detection system in which one of thecomponents is a DNA binding protein which has high affinity forsingle-stranded DNA (ssDNA). To detect the presence of DNA, a sample,optionally pretreated if it contains protein, is denatured to yieldssDNA. The sample is then contacted with a high-affinity,single-stranded DNA binding protein (BP) to form ssDNA-BP complexeswhich may then be detected by means of a label bound to either the ssDNAor the BP. The BP and the ssDNA may both be in solution, at the time ofcontacting, or either of the ssDNA or the BP may be boundnon-diffusively to a solid support prior to the time of contacting. Whenbound to the solid support, the ssDNA or the BP may be bound directly tothe solid support or bound by means of a linker molecule. The method maybe used to detect DNA in, for example, recombinant protein products, orto detect contaminating organisms in a sample.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In accordance with the subject invention, methods and compositions areprovided for detecting the presence of DNA in a sample, particularly ina protein product produced in culture, for example, fermentation. Thepresence of DNA may be detected by the use of high affinity,single-stranded DNA binding proteins.

The method employs single-stranded DNA binding proteins, which may serveto capture and or label single-stranded DNA. The single-stranded DNAbinding protein may be bound to a support or free in solution. When freein solution, the single-stranded DNA binding protein will normallybelabeled. A sample, usually substantially free of protein, is contactedwith a support which binds DNA non-specifically or has, non-diffusivelybound to the support, a protein which binds single-stranded DNAspecifically.

The single-stranded DNA binding protein (BP) may be from any source,either eukaryotic or prokaryotic, and may include single-stranded DNAbinding proteins, topoisomerases, and DNA unwinding proteins. Ofparticular interest are single-stranded DNA binding protein from E. coli(SSB), T4 gene 32 protein and topoisomerase I from Micrococcus luteusand E. coli. Methods of isolation includa affinity chromatography asdescribed in Lohman et al., Biochemistry (1986) 25:21-25. BP is alsocommercially available, for example, from United States BiochemicalCorporation, Cleveland, Ohio.

The DNA binding protein is characterized as having high affinity forsingle-stranded DNA (ssDNA), at least 10⁵ M⁻¹, usually in the range ofabout 10⁸ -10¹⁰ M⁻¹ when the ssDNA is at least 100 nucleotides long.Alternatively, the protein may be characterized as a single-stranded DNAbinding protein requiring a concentration of greater than about 0.4Msodium chloride (or other monovalent salt providing comparable ionicstrength) for elution from ssDNA-cellulose or ssDNA-sepharose. Generallythe concentration for elution is greater than about 0.6M sodiumchloride, and preferably greater than about 1.0M sodium chloride. Whendetermining the affinity, an aqueous buffer, pH 6 to 9 at 25° C. shouldbe used. No detergent, denaturant (for example, urea, guanidiumchloride), chaotropic agent or organic solvent should be present in thebuffer.

The BP can be used unbound to any other component, and/or it can benon-diffusively bound, covalently or adsorptively, to a solid supporteither directly or through a linker molecule or covalently to a label.When a linker molecule is used, the BP is bound to one half of aspecific binding pair, the other half being the linker molecule.Examples of specific binding pairs include biotin and anti-biotin;avidin and streptavidin.

For immobilization of BP, solid support materials may be employed. Thusthe solid support may include filter membranes, preferably Immobilon® ornitrocellule se. For nitrocellulose membranes, the pore size of themembrane is less than 5 μ, preferably less than 1 μ; and is usuallygreater than 0.05 μ, preferably greater than 0.1 μ. Conventionalprocedures as appropriate are used for non-diffusive binding of the BPto the solid support. Methods include covalent binding by reaction withcarbonyl or imidazole groups, or other active groups present on themembrane, or noncovalently by adsorption to the membrane.

The solid support material may also include chromatographic bedmaterials, monodisperse latex particles, including those based onstyrene, chemically-modified styrene, propylene, methacrylate,butadiene, acrylonitrile or other monomers; polyglutaraldehydemicrospheres (e.g., as manufactured by Polysciences, Inc.), nylon beads,chemically-modified nylon beads, oxirane acrylic beads such as Eupergit®(Rohm Pharma, Darmstadt, W. Germany); copolymers of acrylic ester andacrylic amide. Methods of binding BP to these materials include thefollowing: BP may be covalently bound to an activated chromatographicresin having reactive groups capable of forming covalent bonds withproteins, such as CNBr-activated Sepharose-4B, CNBr-activated 4% Agaroseor CNBr-activated Sepharose-6MB (Pharmacia P-L Biochemicals; Piscataway,N.J.), or other resin, such as cellulose, by conventional means. BP maybe bound to polystyrene beads by non-specific adsorption. BP may also bebound covalently to polystyrene beads containing carboxyl or aminofunctional groups (Polysciences, Inc.; Warrington, Pa.) by conventionalmeans.

The DNA, generally denatured to single-stranded DNA (ssDNA), can bebound non-diffusively to a solid support, either absorptively orcovalently, either directly or through a linker molecule. For absorptivebinding, the solid support can be a membrane such as nitrocellulose. Thesample containing the ssDNA is preferably filtered onto the membrane. Tofacilitate binding to the membrane, the salt concentration of the sampleis generally greater than 50 mM sodium chloride, preferably greater than100 mM sodium chloride, or other salt providing similar ionic strengthThe ssDNA is then fixed on the membrane by, for example; baking themembrane at between 75° C. and 100° C., preferably at 80° C. for atleast 30 minutes, usually for at least 1 hour, and preferably no morethan 6 hours; or by washing the membrane with ethanol. When the DNA isbound to the solid support through a linker molecule, the linkermolecule may be any molecule which has high affinity for DNA, such as anantibody to DNA, a BP, or the like. In a preferred method, the linkermolecule comprises a conjugate of biotin and anti-DNA or biotin and BPbound non-diffusively to the solid support through avidin.

The solid support can also be positively-charged nylon, such as beads ormembranes (for example Nytran® (Schleicher and Schull, Inc.; Keene,N.H.), GeneScreen Plus™ (duPont Company; Boston, Mass.), Zeta-Probe®(BioRad Labs; Pinole, Calif.), Bio-Trace™ (Gelman Sciences, Inc.; AnnArbor, Mich.), Bio-dyne Be (Pall Biosupport; Glen Cove, N.Y.), andGenatran™ (Plasco, Inc.; Woburn, Mass.)). The ssDNA can be selectivelynon-diffusively bound to the positively-charged nylon by incubating thenylon in a buffer at between pH 6 and 9 comprising an appropriate saltconcentration and/or non-ionic detergent. The appropriate saltconcentration is preferably less than 1M sodium chloride (or other saltproviding similar ionic strength), and preferably less than 0.6M sodiumchloride. Examples of non-ionic detergents which can be used includeTween-20® or Triton X-100® at a concentration of 0.1-5.0% v/v.

The sample may be any sample in which it is desired to detect DNA whenit is present at a low concentration. The sample may be a solid or aliquid, such as a proteinaceous lyophilized composition or aqueousmedium. Samples can include proteins made by recombinant DNA methods,for example, tissue plasminogen activator, insulin, growth hormone,interleukin 2, and interferons; monoclonal antibodies prepared fortherapeutic purposes; water for use in procedures requiring absolutepurity. The DNA may be in the form of naked DNA, either double-strandedor singlestranded, or it may be in the form of a whole cell, eitherprokaryotic or eukaryotic.

The method for carrying out the subject invention generally is asfollows, although other variations are within the scope of theinvention. If the DNA is contained in whole cells such as bacteria oryeast cells, the cells can be lysed by exposure to lytic conditions suchas treatment with sodium hydroxide, chaotropic agents, such as potassiumthiocyanate, and the like. If the sample contains protein, the proteinis optionally removed As necessary, the DNA is then denatured to ssDNA.

To detect the DNA in the sample, a number of methods may be used, whichinclude the following. The sample can be combined in an assay mediumwith BP to form DNA-BP complexes. The BP may be in solution or bound toa solid support The assay medium is any convenient buffer which willfacilitate binding of the various assay components. The ssONA is thenbound non-diffusively to a solid support, for example via binding to BPbound diffusively or non-diffusively to the solid support. The complexesare then freed of any unbound sample and BP, and the complexes detectedby means of the label as indicative of the presence of DNA in thesample. If the solid support does not contain non-diffusively bound BP,the ssDNA may be fixed on the solid support by, for example, baking,treatment with ethanol, or other convenient means. BP is then added tothe solid support where it binds specifically to the ssDNA. Whether anyDNA is present in the sample is determined by detecting ssDNA-BPcomplexes on the solid support by means of a label, generally bound toeither the BP or the ssDNA.

Instead of being bound to the solid support directly the BP may be boundindirectly through a specific binding pair member label bound to the BPwhich in turn is bound to the solid support via a specific binding pairmember, complementary to the specific binding pair member label, whichis immobilized on the solid support. Examples of specific binding pairsinclude biotin and avidin; biotin and streptavidin; and biotin andantibiotin. Following contact of a sample comprising ssDNA with thesolid support, a second BP comprising a detectable label is then addedand DNA presence determined by means of the detectable label.

When using the assay system where the BP is bound indirectly to thesolid support, the components of the assay system may be combinedtogether on a solid support, but preferably are added sequentially,including a step after addition of each component to remove any unboundassay components. For example, BP labeled with a specific binding pairmember (sbpm) such as biotin is contacted with a solid supportcomprising a complementary specific binding pair member (s'bpm) such asantibiotin and the solid support washed to remove any unbound specificbinding pair members. Sample containing ssDNA is then added where itbinds to the BP bound to the solid support. BP comprising a detectablelabel (d1) such as an enzyme is then added to the solid support where itbinds to the ssDNA. Thus the solid support comprises(d1-BP)-ssDNA-(BP-sbpm)-s'bpm complexes. The solid support is then freedof any uncomplexed dl-BP and the presence of DNA determined by means ofthe detectable label.

When it is desired to determine the concentration of DNA present, inaddition to the sample, usually there will be at least one standardsolution tested; there will be at least one background solutioncontaining no DNA; and there will be at least one reference solutioncontaining a known amount of DNA; all of which are treated identicallyto the sample containing an unknown concentration of DNA. The amount oflabe1 detectable in the background solution is subtracted from theamount of labe1 detectable in the reference solution and the unknownsample. The adjusted values for the reference solution and the unknownsample are then related to determine the amount of DNA present in thesample.

The following are general methods for carrying out the above steps. Themethod of the present invention can be used for the detection of DNA ineither the presence or absence of protein. When protein is present, anadditional step to deproteinize the sample is desirable. Anyconventional means for deproteinization can be used (for example, phenolextraction) which does not adversely affect the integrity of the DNA. Ifthe protein has known characteristics, the sample may be deproteinizedby ion-exchange column chromatography (for example, DEAE-cellulose,phosphocellulose, sulfonic gel), hydroxyapatite (the single-stranded anddouble-stranded DNA may be separated from protein by elution withdifferential salt concentrations), gel filtration, and affinitychromatography.

Affinity chromatography may be used to remove the protein directly fromthe sample, e.g., using immobilized mouse immunoglobulin raised againstthe protein to be removed, or the sample may be deproteinized usingimmobilized BP to bind the DNA in the sample. The DNA can then be elutedfrom the BP using a high-salt concentration, usually greater than 1M,preferably greater than 2M, and the eluant used directly in thedetection assay after adjusting the salt concentration. The final saltconcentration varies depending upon the protocol used. For example,where the solid support is a positively charged nylon membrane or amembrane comprising a linker molecule, the salt concentration isadjusted to isotonic. For detection of DNA in a monoclonal antibodysample, the monoclonal antibody may be removed using protein A bound toa solid support.

Other methods of deproteinizing the sample include mixing the samplewith a suspension of glass particles in the presence of a highconcentration of sodium iodide. Any DNA present in the sample isnon-diffusively bound by the glass particles. The glass particles areisolated, and the DNA recovered from the particles by treatment withwater or PBS. The glass particles may include finely ground glass beads,or preferably a composition comprising Glassmilk™ as supplied by BIO101, Inc., La Jolla, Calif.

Another method which can be used to deproteinize the sample is admixingthe protein-containing sample with a proteolytic enzyme compositioncomprising, for example, at least one of the enzymes pronase orproteinase K. Following the enzymatic treatment, hydrolyzed product isoptionally removed, for example, by gel filtration or by centrifugationthrough a membrane with a low molecular weight cutoff (approximately10,000 or 30,000 as supplied by Centricon-10, Centricon-30; Amicon,Danvers, Mass.); use of a Millipore low-volume ultrafiltration devicewith a low molecular weight cutoff (approximately 10,000 or 30,000).After any protein present is digested, removed, or digested plusremoved, the DNA is denatured to ssDNA. Methods used to denature the DNAinclude heating at about 90°-100° C. or treatment with sodium hydroxide(pH 13.0). After rapid chilling (to prevent the DNA from reannealing) orneutralization (using for example, ammonium acetate or Tris buffer), thessDNA is contacted with a solid support.

If the solid support is a membrane such as nitrocellulose orpositively-charged nylon, the sample is generally filtered using amanifold filtration device. However, if the solid support is, forexample, positively-charged nylon beads, the beads can be incubateddirectly in the sample. When the solid support has BP immobilized on itssurface, the ssDNA binds to the BP to form BP-ssDNA complexes. If thesolid support does not contain BP, the DNA is fixed on the solid supportby baking, or treatment with ethanol. This step can be omitted when apositively-charged nylon membrane is used.

Non-specific binding sites on nitrocellulose or positively-charged nyloncan be blocked by incubation of the membrane or beads with ahighconcentration protein solution such as about 1 to 10% bovine serumalbumin (BSA), non-fat dry milk solution and the like. For thepositively-charged nylon, the non-specific binding sites additionallycan be blocked by washing with a non-ionic detergent solution, such asTween-20® or Triton X-100®, usually 0.1-5.0%.

The solid support comprising non-diffusively bound ssDNA is thenincubated with labeled BP to form BP-ssDNA complexes. When the solidsupport is a nitrocellulose or positively-charged nylon membrane, buffer(pH 6-9) containing BP (generally about 0.3 μg/ml) is added to themembrane. The buffer is generally at room temperature and containssodium chloride, preferably 0.01-0.3M, and in addition, for thepositively-charged nylon, contains a non-ionic detergent such asTween-20® or Triton X-100®, preferably 0.1-5.0% v/v.

Any BP-ssDNA complexes can be detected by means of a label attached toeither the BP or the ssDNA, the label preferably being attached to theBP. An exception is when the BP is pre-attached to the solid support,when the DNA is preferably labeled. The BP can be covalently labeled ina number of ways. The label can be an enzyme, for example, alkalinephosphatase, β-D-galactosidase, glucose-6-phosphate dehydrogenase,glucose oxidase, horseradish peroxidase, β-lactamase, urease; aradionuclide, such as ¹²⁵ I; a chemiluminescent or fluorescent compound,such as fluorescein isocyanate; a hapten such as biotin, and the like;or any other label which provides a detectable signal. When the label isan enzyme such as urease, which contains at least one free, accessible,non-essential cysteine residue. BP can be coupled to the enzyme, forexample, as described by Blakely et al., J. Mol. Catalysis (1984)23:263-292). Other enzymes which can be coupled in this way includeβ-D-galactosidase.

Alternatively, an enzyme label can be thiolated and then conjugated tothe BP. Methods for attaching labels to proteins are described in detailin the scientific literature. See for example Healey et al., ClinicaChimica Acta (1983) 134:51-58; Ishikawa et al., J. Immunoassay (1983)4:209-327; and Tijssen, Practice and Theory of Enzyme Immunoassays(1985) 259-63, Elsevier Science Publishers [Amsterdam].

When the label on BP is an enzyme, it is convenient to use a mercaptancoupling member corvalently bounded to the BP for linking mercaptangroups on the enzyme to the BP. In the case of SSB the number ofmaleimide molecules for mercaptan linking bound per SSB molecule isusually about 1 to 3 maleimide molecules/SSB, preferably 1.8 maleimidemolecule/SSB. Generally this results in an SSB-enzyme conjugatecomprising 1 enzyme molecule per SSB molecule. When the label is ahaptan such as biotin, the number of hapten molecules bound per SSBmolecule is usually 1 to 5, preferably 3 to 4.

The DNA to be detected can be labeled, rather than the BP. The label mayinclude a radionuclide, fluorophore, or a hapten such as digoxin orbiotin and the like. The label can be introduced to the DNA by anystandard enzymatic reaction such as nick translation, or by terminallabeling, with ³ H, ¹⁴ C ³² P, or biotin-labeled deoxynucleotidetriphosphates (dNTP). The labeled DNA is then denatured to ssDNA byalkali or heat.

Alternatively, the DNA can be labeled with a reagent such as isopsoralenwhich binds to doublestranded DNA. 3H-isopsoralen or biotin-isopsoralenis available from HRI Research, Inc., Berkeley, Calif. The isopsoralenreagent is bound to DNA by mixing it with a sample, followed byphotoirradiation at 340-380 nm. When the label used is isopsoralen, itis unnecessary to denature further the labeled DNA, asisopsoralen-labeled DNA is recognized by BP without any additionaldenaturing step.

Methods of detecting BP-ssDNA complexes will depend on the type of labelused as well as the sensitivity required. When the label is an enzyme,the disappearance of substrate or appearance of reaction product may bemeasured spectrophotometrically following substrate addition If theenzyme is, for example, urease, an indicator dye such as cresol red maybe used to monitor the change in pH in the sample following addition ofenzyme substrate. The change in optical density or the visual intensityof the color change is then correlated with the DNA content of thesample by comparison with at least one identically treated referencesolution containing a known concentration of DNA. Alternatively, any DNApresent may be detected by measuring the amount of pH or potentialchange with a photoresponsive device such as that described in U.S. Pat.No. 4,591,550. Other methods of detecting BP-ssDNA complexes in thesample may include quantitating the amount of radioactivity, when thelabel is a radionuclide. When the label is a hapten such as biotin, thelabel can be detected by, for example, the use of enzyme-labeled avidin,streptavidin or antibiotin.

Various protocols may be used. For example, a sample suspected ofcontaining an organism or free DNA may be lysed in the former case, andin both cases denatured to provide ssDNA. The sample may then becontacted with a positively charged nylon membrane or a membrane towhich a high affinity ssDNA binding protein is conjugated. Any DNA maythen be detected by employing an ssDNA binding protein conjugate bondedto a detectable label.

For convenience, the reagents are frequently provided in kits, wherethey may be present in conjunction with buffer, stabilizers, excipientsand the like. The kit may also include any additional reagents necessaryfor the preparation of labeled BP or ssDNA and the detection of thelabeled BP-ssDNA complexes in the performance of the assay. Where thereagents include BP, it may be provided labeled or unlabeled. Whenunlabeled, it may also be provided bound non-diffusively to a solidsupport.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1 Preparation of SSB-Enzyme Conjugates A.Preparation of SSB Urease Conjugate Using m-MaleimidobenzoylN-hydroxysuccinimide Ester (MBS)

Single-stranded DNA binding protein from E. coli (SSB) was obtained fromUnited States Biochemical Corp. Cleveland, Ohio. It was coupled to thecrosslinking agent MBS as follows. One hundred μ1 of 0.25% MBS indimethylformamide (DMF) was added to 2.0 ml containing 2 mg of SSB in0.1M phosphate buffer, pH 6.8. The mixture was stirred gently at roomtemperature for 30 min then separated on a Sephadex G-25. The elutionbuffer was 0.1M phosphate, pH 6.8. Fractions were monitored by UVabsorbance at 280 nm. The first peak eluted from the column oontainedSSB coupled to MBS. The peak fractions (3 ml) were combined with 4 ml ofurease (20 mg) in 0.1M phosphate buffer, pH 6.8. The mixture was stirredfor 20 min at room temperature. The reaction was then stopped by theaddition of 1.75 ml of 500 mM sodium sulfite, in 0.1M sodium phosphatebuffer, pH 6.8, containing 10 mM dithiothreitol (DTT). The conjugateformed was either used directly in the assay or was separated fromunconjugated enzyme by gel filtration chromatography. The purifiedenzyme conjugate was then diluted 1:1 (v/v) with glycerol. BSA was addedto 0.25% (w/v). The conjugate was stored at 2°-8° C.

B. Preparation of SSB Horseradish Peroxidase (HRP) Conjugate Usingm-maleimidobenzoyl N-hydroxysuccinimide ester (MBS)

HRP (Boehringer-Mannheim, La Jolla, Calif.) was thiolated withN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Pierce, Rockville,Ill.) by combining 16 mg HRP in 0.1M potassium phosphate buffer, 0.2Msodium chloride, pH 7.5 with 435 μg SPDP in 25 μl dimethylformamide(DMF), and incubating for 30 min at room temperature. Unreacted SPDP wasremoved by chromatography on Sephadex G-25 and eluted with 0.1Mpotassium phosphate buffer containing 0.2M sodium chloride, pH 7.0.

Dithiopyridine groups on HRP were dehlocked hy adding 25 mM DTT for 30min, then removing the DTT and the 2-thiopyridine formed by G-25separation in PBS.

Maleimido-SSB was formed by adding 25 μof a 0.25% solution of MBS in DMFto 0.55 mg of SSB in 0.5 ml of 0.1M sodium phosphate buffer, andstirring for 30 min at room temperature. Maleimido-SSB was purified onSephadex G-25, then condensed with 15 mg of thiolated HRP by combiningthe two solutions and reacting for 20 min at room temperature. Thereaction was stopped by addition of 12.5 μl of 100 mM 2-mercaptoethanol.The SSB-HRP conjugate solution was made 10% (v/v) in glycerol and storedat 4° C.

C reparation of SSB-Biotin Conjugate.

One mg of SSB in PBSE (150 mM sodium phosphate pH 7.0, NaCl 50 mM, and 1mM EDTA) Was mixed with 50 μg of biotinamidocaproateN-hydroxysuccinimide ester in 20 μl of DMF for 2 hrs at room temperaturewith stirring. Unreacted biotin reagent was removed by passage over aSephadex G-25 column.

D. Preparation of Anti-DNA-Urease Conjugate

One mg of purified anti-DNA monoclonal antibody, clone 4H2 (obtainedfrom Dr. Richard Weisbart at the Sepulveda Veterans AdministrationHospital, Los Angeles) in 1 ml PBSE was reacted with 125 μg of4-(N-maleimido methyl) cyclohexane-1-carboxylic acid Nhydroxysuccinimideester (SMCC) in 50 μl of DMF. Unreacted SMCC was removed by passage overa Sephadex G-25 column eluted with PBSE. The number of maleimide groupsper antibody was 4. Urease (20 mg) was then added to themaleimide-antibody and incubated for 4 hr at 4° C. Unreacted maleimidegroups were then blocked by addition of 2-mercaptoethanol to aconcentration of 2 mM.

Conjugate was purified by gel filtration chromatography on a 1.5 cm×70cm column of Sephacryl S-400 HR (Pharmacia, Piscataway, N.J.) in abuffer composed of 50 mM sodium sulfite, 20 mM sodium dihydrogenphosphate, 200 mM sodium chloride, 1 mM EDTA, 2 mM dithiothreitol and0.1% Tween-20. The entire protein peak eluting ahead of the free ureasepeak was combined. 0.05% BSA was added and the conjugate solution wasstored at 4° C. in sealed containers under argon gas.

E. Preparation of Biotin-Anti-DNA Conjugate

One mg of purified anti-DNA monoclonal antibody obtained from R.Weisbart in PBSE was added to 50 μg of biotin amidocaproate-N-hydroxysuccinimide ester in 20 μl DMF and stirred for 2 hrsat room temperature. Unreacted biotin reagent was removed by passageover a Sephadex G-25 column. Purified anti-DNA-biotin was stored at 4°C.

EXAMPLE 2 Binding of SSB to Solid Supports A. Immobilization of SSB onan Immobilon™ Membrane

Immobilon™ membrane contains carbonyl imidazole groups which bind toepsilon amino groups on lysine or arginine. SSB was immobilized on a0.65 μ Immobilon™ membrane (Millipore; Bedford, Mass.) by soaking themembrane in phosphate buffered saline containing 0.2 mg/ml SSB at roomtemperature for 1 hour (10 cc protein solution/ 100 cm² membrane). TheSSB solution was removed by decanting. Any remaining active carbonylimidazole groups on the membrane were quenched with 0.1M ethanolamine(pH 9.5) at room temperature overnight. The membrane was then washedsuccessively in phosphate buffered saline, distilled water, andpolyvinylalcohol (15 min each wash), and then dried at 65° C. for 5 min.The dried membrane was then ready for use in the detection system. Fourhundred μl of a sample containing single-stranded ³² P-labeled DNA (from10-1000 pg) in a 10 mg/ml BSA aqueous solution was filtered through theSSB-Immobilon™ membrane (filtration time of 10 min). Seventy percent ofthe ³² P counts were captured on the membrane when the SSB concentrationwas 0.2 mg/ml. Increasing the SSB concentration used in theimmobilization procedure to 1 mg/ml improved the DNA capture to 80%.

B. Immobilization of SSB on Nitrocellulose Membranes

SSB was adsorbed to a nitrocellulose membrane (0.45 μ pore size,Schleicher and Schuell) by soaking the membrane in PBS containing 50μg/ml SSB at 4° C. overnight. Non-specific binding sites on the membranewere blocked with 10 mg/ml BSA at room temperature for 1 hour. When 400μl of samples containing 10 mg/ml BSA and ³² P-labeled single-strandedDNA were filtered through the membrane, 36% of the ³² P counts werecaptured on the nitrocellulose membrane.

EXAMPLE 3 Detection of Pure DNA in a Sample A. Visual Determination

Samples containing pure calf thymus DNA (Sigma Chemical Co., St. Louis,Mo.) from 0-100 pg/sample (in 10 mM sodium phosphate, pH 7.0, 0.15Msodium chloride, 1 mM EDTA) were denatured to single-stranded DNA byheating at 100° C. for 10 min followed by rapid chilling. The denaturedsamples were filtered through 0.45 μ nitrocellulose membrane (Schleicherand Schuell; Keene, N.H.) using a manifold filtration device. Themembranes were then baked at 80° C. for 1 hour to fix the DNA.SSB-urease conjugate, prepared as in Example IA and used without furtherpurificatlon, was diluted to 0.3 μg/ml in 2% bovine serum albumin (BSA),2% Ficoll, 2% polyvinylpyrrolidone, 10 mM sodium phosphate, 40 mM sodiumchloride, 2 mM EDTA, pH 7.5 was added to the membrane. The membrane wasincubated with the SSBurease conjugate for 1 hour at room temperature ina Petri dish in a sufficient volume of conjugate to cover the membrane.The membrane was then washed three times with 0.15M sodium chloride, 1mM EDTA (pH 6) to remove any non-specifically bound conjugate. Any DNApresent was then detected by the addition of enzyme substrate (100 mMurea, 0.15M sodium chloride, 1 mM sodium phosphate, pH 6, 0.5 mM cresolred). The change in pH due to the urease reaction resulted in a colorchange of the cresol red from orange to purple-red. The visual intensityof the purple-red spot was then correlated with the DNA content of thesample by determining the relative size of the colored spots on themembrane and the intensity of the color.

                  TABLE 1                                                         ______________________________________                                        COLORIMETRIC DETECTION OF DNA                                                 Pure DNA      Intensity of                                                    (pg/sample)   Color at 3 min                                                  ______________________________________                                         0            -                                                               10            +                                                               20            ++                                                              50            +++                                                             100           ++++                                                            ______________________________________                                    

B Biosensor pH determination

Any DNA present on membranes prepared as described above can also bedetected by measuring the amount of pH change following addition ofenzyme substrate, using a photoresponsive device (see for example U.S.Pat. No. 4,591,550). An enzyme substrate mixture containing 1 mM sodiumphosphate, 0.05% Tween-20, mM urea, pH 6 was added to the membranes. Thechange in pH due to the urease reaction resulted in a change in thesignal of the photoresponsive device. The change in the signal (inμvolt/ sec) was then correlated with the DNA content of the sample.

                  TABLE 2                                                         ______________________________________                                        DETECTION OF DNA BY BIOSENSOR                                                 pH DETERMINATION                                                              DNA (pg/sample)  μvolt/sec                                                 ______________________________________                                         0                64                                                          12               107                                                          25               153                                                          50               268                                                          100              341                                                          ______________________________________                                    

EXAMPLE 4 Preparation of Solid Supports for Specific Caoture of DNA A.Preparation of Anti-Biotin Membrane

1. Nitrocellulose

Anti-biotin (Sigma) was dissolved in PBS at a concentration of 0.25mg/ml. A nitrocellulose sheet (0.8 μ) (Schleicher and Schuell) waswetted with DNA-free water, then incubated in the anti-biotin mixture(0.2 ml/cm² membrane) with gentle rocking membrane for 15 min at roomtemperature, then overnight at 4° C. The anti-biotin mixture was thenpoured off and the nitrocellulose rinsed briefly with pBS (0.2 ml/cm²).The nitrocellulose was then incubated with 0.1% (w/v) glutaraldehyde inPBS for 15 min. The nitrocellulose was then washed successively withPBS, then DNA-free water, both at 0.2 ml/cm². The nitrocellulose wasthen wetted with 0.2% (w/w) polyvinyl alcohol for 10 min at 0.2 ml/cm².The nitrocellulose was then baked for 10 min at 60° C.

2. Cellulose acetate

An anti-biotin membrane was prepared as described in Section 3A.1,above, substituting a cellulose acetate membrane (Schleicher andSchuell, 1.2 pore size), except that the wash with PBS prior to theglutaraldehyde/PBS incubation was omitted.

EXAMPLE 5 Detection of DNA in Samples Containing Protein withNitrocellulose Membrane A. Protein removal by glass beads

Four hundred μl of sodium iodide (6M) were added to 200 μl of samples,each containing 2 mg of BSA and 100, 50, 25, or 0 pg calf thymus DNA.Two μl of Glassmilk™ were added and the mixture incubated for 10 min atroom temperature. The Glassmilk™/DNA complex was pelleted bycentrifugation for 10 sec in a microcentrifuge. The pellet was washedwith 150 μl 20 mM Tris buffer, containing 200 mM sodium chloride, 2 mMEDTA in 55% methanol. The wash procedure was repeated once. After theDNA was eluted with 400 μl phosphate buffered saline, each sample washeated at 100° C. for 10 min to denature the DNA then rapidly chilled.The sample was filtered onto nitrocellulose membranes. DNA was detectedusing the visual determination procedure described in Example 3.A.

                  TABLE 4                                                         ______________________________________                                        DETECTION OF PROTEIN-CONTAINING SAMPLES                                       DNA             Intensity of                                                  DNA (pg/sample) Color at 3 min                                                ______________________________________                                         0              slight positive                                               25              ++                                                            50              +++                                                           100             ++++                                                          ______________________________________                                    

B. Protease digestion of protein

Proteinase K and dithiothreitol were added (final concentration 100μg/ml and 50 mM, respectively) to 100 μl of samples each containing 1 mgBSA and 100, 50, 25, 12, or 0 pg of DNA in phosphate buffered saline.The mixture was incubated at 55° C. for 2 hours to digest the protein.After digestion, all samples were heated at 100° C. for 5 min toinactivate proteinase K and denature DNA to single-stranded DNA. Controlsamples containing a matching amount of DNA but no protein weredenatured at the same time. Each sample, after rapid chilling to preventreannealing of the DNA, was filtered through a nitrocellulose membrane.Visual determination procedures were carried out as described in Example3.A to detect the presence of DNA.

                  TABLE 5                                                         ______________________________________                                        DETECTION OF PROTEINASE-DlGESTED,                                             PROTEIN-CONTAINING SAMPLES                                                                    Intensity                                                     DNA             With     Without                                              (pg/sample)     Protein  Protein                                              ______________________________________                                         0              -        -                                                    12              +        +                                                    25              ++       ++                                                   50              +++      +++                                                  100             ++++     ++++                                                 ______________________________________                                    

EXAMPLE 6 Detection of DNA in Sample Containing Protein by Adsorption ofthe DNA to Positively Charged Nylon

7.5 μl of proteinase K (2 mg/ml) were then added to 150 μl solution ofporcine insulin (Cal Biochem, La Jolla, Calif.) 10 mg/ml in 10 mMTRISHCl 1 mM EDTA, pH 8.7, containing 0, 5, 10, 20, or 40 pg ofdouble-stranded calf thymus DNA. The samples were incubated overnight at55° C., boiled at 100° C. for 5 min, then chilled on ice. The digestedsamples were then filtered through a positively charged nylon membrane(Genatran™, 6 cm×8 cm obtained from Plasco, Inc., Woburn, Mass.) at arate of about 100 μl/min. DNA was detected by incubating the membranewith 0.2-0.5 ml/cm² SSB-HRP conjugate (150 ng/ml in 50 mM sodiumphosphate, pH 7.4; 150 mM NaCl; 2 mM EDTA, 0.1 mg/ml BSA; 5% Triton-X100®) for 40 min at room temperature in a petri dish. The membrane wasthen washed three times by incubating the membrane in PBS containing 1Murea and 1% dextran sulfate for 3 min each wash to remove anynon-specifically bound SSB-HRP. The membrane was then washed withdistilled water, incubated for 10 min in 10 mM sodium citrate buffercontaining 10 mM EDTA, 0.1 mg/ml tetramethyl benzidine and 0.001%hydrogen peroxide, pH 5. The visual intensity of the blue spots whichdeveloped on the membrane was determined subjectively, then correlatedwith the DNA content of the original sample. The results were as shownin Table 6.

                  TABLE 6                                                         ______________________________________                                        VISUAL DETECTION OF DNA USING SSB-HRP                                         pg of DNA in   Intensity                                                      Insulin Sample of Color                                                       ______________________________________                                        40             ++++                                                           20             ++                                                             10             +                                                               5             slight positive                                                 0             -                                                              ______________________________________                                    

EXAMPLE 7 Detection of DNA Using Specific Capture of Sample DNA on anAnti-Biotin Membrane A. Purification of SSB-Urease Conjugate

In this example, SSB-urease was further purified to remove unconjugatedurease by gel filtration chromatography of the SSB-urease on a column of1 cm×30 cm of Superose 6 (Pharmacia, Piscataway, N.J.) in a buffercontaining 50 mM sodium sulfite, 20 mM sodium dihydrogen phosphate, 200mM sodium chloride, 1 mM EDTA, 2 mM dithiothreitol, 0.1% Tween-20 pH7.00. The protein peak eluting before the unconjugated urease peak wascollected and combined with glycrol 1:1 (v:v) and stored at -20° C.

B. Biosensor Detection of DNA in Buffer with SSB-Urease Conjugate(Sequential Addition of Reagents)

Anti-biotin-coated nitrocellulose membranes prepared as described inExample 4A1, were coated with biotin-anti-DNA by filtering 200 μl ofbiotin-anti-DNA (see Example 1E), in 300 ng/ml in 10 mM TRIS-HClcontaining 1% BSA, 1 mM EDTA through the antibiotin membrane over aperiod of about 4 min. The membranes were then washed with 300 μl PBS.200 μl of the samples containing DNA (0, 12, 25, 50 pg/200 μl DNA inpBS) were filtered over a period of about 4 min. The filter was againwashed with 300 μl of PBS. Over about 4 min, 200 μl of SSB-ureaseconjugate, purified as described in Example 7A, (100 ng/ml diluted in 2%BSA, 2% Ficolle, 2% polyvinylpyrrolidone, 10 mM sodium phosphate, 40 mMNaCl 2 mM EDTA, pH 7.5) was filtered through the membrane. The membranewas then washed 3 times with 1 ml of 1 mM sodium acetate, 0.1M NaCI,0.05% Tween-20, pH 5. The membrane was added to substrate solution(acetate wash buffer containing 100 mM urea) and the rate of productformation measured as described above in Example 3B. The rate of productformation was a function of the concentration of DNA in the sample, asshown in Table 7.

                  TABLE 7                                                         ______________________________________                                        BIOSENSOR DETECTION OF DNA                                                    USING SSB-UREASE CONJUGATE                                                           Amount of                                                                             Rate                                                                  DNA (pg)                                                                              (μV/sec)                                                    ______________________________________                                               50      546                                                                   25      300                                                                   12      183                                                                    0      120                                                            ______________________________________                                    

C. Titration of Biotin-Anti-DNA for a Simultaneous-Incubation Assay

The effect of simultaneously adding biotin-anti-DNA and SSB-ureaseconjugate purified as described in Example 7A to the antibiotin coatednitrocellulose membrane was assessed as follows:

200 μl of sample (1% BSA in TE with or without 100 pg ssDNA) wasincubated with 200 μl biotin-anti-DNA (40, 81, 162, 325 ng/ml in 1% BSA,TE) and 200 μl SSB-urease conjugate (15 ng/ml in 2% BSA, 2% Ficolle, 2%polyvinylpyrrolidone, 10 mM sodium phosphate, 40 mM NaCl, 2 mM EDTA, pH7.5) at room temperature for 50 min. The mixture was then filteredslowly over a period of about 10 min through an antibiotin coatednitrocellulose membrane (see Example 4.A.1). The membrane was thenWashed 3 times with 1 ml of acetate wash buffer (1 mM sodium acetate,0.1M NaCl, 0.05% Tween-20, pH 5). Substrate solution (acetate washbuffer containing 100 mM urea) was added and the amount of DNA presenton the membrane determined using the biosensor procedure as describedabove in Example 3B. The results, shown in Table 8 below, show that in asimultaneous assay, the ratio of biotin-anti-DNA and SSB-ureaseconjugate is critical, presumably due to competition of both anti-DNAand SSB for DNA.

                  TABLE 8                                                         ______________________________________                                        TITRATION OF BIOTIN-ANTI-DNA THROUGH                                          SIMULTANEOUS-INCUBATION ASSAY                                                                   Rate                                                        Concentration of  (μV/sec)                                                 Biotin-Anti-DNA (ng/ml)                                                                         100 pg DNA No DNA                                           ______________________________________                                        325                308       108                                              162                476       149                                               81               1046       238                                               40               1308       217                                              ______________________________________                                    

Biosensor Detection of DNA in Granulocyte Macrophage Colony StimulatingFactor (GM-CSF) Samples with SSB-HRP Conjugate (Sequential Addition)

300 ∥l of recombinant human GMCSF (Biogen, Cambridge, Mass.), 3.4 mg/mlPBS, was spiked with 0, 5, 10, 50, or 100 pg double-stranded calf thymusDNA. The samples were heated to 100° C. for 5 min to denature the DNA,then cooled to room temperature. Immobilon™ membranes (Millipore,Bedford, Mass.) were coated with goat anti-biotin IgG (Sigma, St Louis,Mo.), as described above for SSB coating on Immobilon™ in Example 2A.200 μl of biotin-labeled anti-DNA (clone 4H2) was filtered through themembrane. 300 μl of the samples containing GMCSF were filtered throughthe membranes and the membranes washed again with 200 μl of PBS. 200 μlof SSB-HRP conjugate (650 ng/ml, diluted in PBS containing 0.1 mg/mlBSA, 2 mM EDTA, 5% Triton-X 100) was filtered through the membraneMembranes were then washed once with PBS containing 5% Triton-X 100, 1Murea, and washed with 0.1M sodium acetate, containing 5% ethanol and 1mM EDTA, pH 5.5. The amount of DNA was then determined by the biosensormethod as described above in U.S. Pat. No. 4,591,550, which disclosureis incorporated herein by reference. The substrate solution comprisedsodium acetate wash buffer containing 250 μM tetramethyl benzidine, 50μM ruthenium, 500 μM hydrogen peroxide, pH 5.5. The change in potentialdue to the HRP redox reaction resulted in a change in the signal of thephotoresponsive device. The results were as shown in Table 9, below.

                  TABLE 9                                                         ______________________________________                                        BIOSENSOR DETECTION OF DNA IN GM-CSF                                          SAMPLES USING SSB-HRP CONJUGATE                                               pg of DNA in 1 mg                                                                              Rate                                                         GMCSF (3 mg/ml)  (μV/sec)                                                  ______________________________________                                         0               -137                                                          5               -201                                                         10               -268                                                         50               -629                                                         100              -1550                                                        ______________________________________                                    

EXAMPLE 9 Detection of Bacteria in a Water Sample

Cultures of gram-negative and gram-positive bacteria are diluted in 100μl HPLC-grade water to 80, 400, 2,000, 10,000/100 μl. For one set ofsamples, the bacteria are lysed and denatured by treatment with 10 μl of3N NaOH, then neutralized with 50 μl of 1M TRISHCl, pH 7.3. A secondidentical set of dilutions was not so treated and serve as a control fornon-specific interaction between the sample and the SSB-HRP conjugate.

Both sets of dilutions are loaded onto a positively charged nylonmembrane (Genatran™) using a dot-blot apparatus (Schleicher andSchuell). Each well of the apparatus is loaded with 165 μl of sample.Standards, consisting of denatured calf thymus DNA diluted in HPLC-gradewater) are also prepared and loaded onto the positively charged nylonmembranes. Standards contain 0, 5, 10, or 50 pg ssDNA. DNA is detectedusing the visual assay as described above in Example 3A using an SSB-HRPconjugate.

The subject methods and compositions provide a rapid and simple meansfor detecting picogram amounts of DNA in a sample by the use of highaffinity single-stranded DNA binding proteins The assay is applicablenot only to pure DNA samples but may also be used with samples whichcontain a significant amount of protein, or for detecting contaminationof a sample with a microorganism.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed:
 1. A method for quantitatively detecting ssDNA in asample, said method comprising:combining, in an assay medium, saidsample and assay components for detecting said ssDNA, said componentscomprising: a high affinity single-tranded DNA binding protein labeledwith a detectable label (d1-BP), a high affinity single-stranded DNAbinding protein labeled with a specific binding pair member (BP-sbpm)and an immobilized complementary specific binding pair membercomplementary to said sbpm-BP (s'bpm), whereby a DNA complex formsbetween any ssDNA in said sample and said binding proteins, and saidBP-sbpm binds to said immobilized s'bpm; and detecting immobilizeddetectable label as indicative of the presence of ssDNA in said sample.2. The method according to claim 1, wherein said combining comprisesadding said sample and said assay components sequentially asfollows:contacting said immobilized s'bpm with said BP-sbpm wherebyimmobilized (BP-sbpm)-s'bpm first complexes are formed; contacting saidfirst complexes with said sample whereby immobilizedssDNA-(BP-sbpm)-s'bpm second complexes are formed; and contacting saidsecond complexes with said BP-d1 whereby immobilized(BP-d1)-ssDNA-(BP-sbpm)-s'bpm third complexes are formed.
 3. The methodaccording to claim 2, further comprising, after each contacting step,freeing said immobilized complex of any unbound assay component.
 4. Themethod according to claim 1, wherein said sample is subjected to DNAdenaturing conditions prior to said combining.
 5. The method accordingto claim 1 further comprising:additionally treating at least onereference solution containing a known amount of DNA according to themethod of claim 6; and relating the signal from the detectable labeldetected in said sample to the signal detected from said detectablelabel in said reference solution to determine the amount of DNA presentin said sample.
 6. The method according to claim 1, wherein saiddetectable label is an enzyme.
 7. The method according to claim 1,wherein said specific binding pair members are biotin and avidin; biotinand streptavidin; or biotin and antibiotin.
 8. A method forquantitatively detecting an organism in a sample, said methodcomprising:treating said sample under lysing and denaturing conditionsto provide ssDNA in a lysate; contacting said lysate with a positivelycharged nylon membrane or a nitrocellulose membrane, whereby ssDNAbecomes bound to said membrane; contacting said member with a highaffinity ssDNA bindign protein conjugated to a detectable label to formcomplexes with any ssDNA bound to said membrane; and detecting thepresence of said detectable label bound to said membrane as indicativeof an organism in said sample.
 9. The method according to claim 8,wherein said label is an enzyme.
 10. The method according to claim 8,wherein said organism is a bacterium, a yeast cell or a virus.
 11. Themethod according to claim 1, wherein the high affinity single-strandedDNA binding protein has an affinity for single-stranded DNA of at leastabout 10⁵ M⁻¹.
 12. The method according to claim 1, wherein the highaffinity single-stranded DNA binding protein has an affinity forsingle-stranded DNA of abnout 10⁸ to about 10¹⁰ M⁻¹.
 13. The methodaccording to claim 1, wherein the high affinity single-stranded DNAbinding protein has a single-stranded DNA binding affinity such that thehigh affinity single-stranded DNA binding protien may be eluted fromsingle-stranded DNA-cellulose or single-stranded DNA-Sepharose by asodium chloride concentration of greater than about 0.4M.
 14. The methodaccording to any one of claim 1, wherein the high affinitysingle-stranded DNA binding protein has a single-stranded DNA bindingaffinity such that the high affinity single-stranded DNA binding proteinmay be eluted from single-stranded DNA-cellulose or single-strandedDNA-Sepharose by a sodium chloride concentration of greater than about0.6M.
 15. The method according to claim 8, wherein the high affinitysingle-stranded DNA binding protein has an affinity for single-strandedDNA of at least about 10⁵ M¹.
 16. The method according to claim 8,wherein the high affinity single-stranded DNA binding protein has anaffinity for single-stranded DNA of about 10⁹ to about 10¹⁰ M⁻¹.
 17. Themethod according to claim 8, wherein the high affinity single-strandedDNA binding protein has a single-stranded DNA binding affinity such thatthe high affinity single-stranded DNA binding protein may be eluted fromsingle-stranded DNA-cellulose or single-stranded DNA-Sepharose by asodium chloride concentration of greater than about 0.4M.
 18. The methodaccording to claim 8, wherein the high affinity single-stranded DNAbinding protein has a single-stranded DNA binding affinity such that thehigh affinity single-stranded DNA binding protein may be eluted fromsingle-stranded DNA-cellulose or single-stranded DNA-Sepharose by asodium chloride concentration of greater than about 0.6M.